ABSTRACT. Purpose: To study the
stability of levamisole oral solutions (25 mg/mL) prepared from powder and
tablets stored at 4 ±3°C
and 23 ±2°C
in amber glass prescription bottles. Methods: Levamisole 25 mg/mL
solutions were prepared from commercially available 50-mg tablets or from pure
powder in sterile water. Levamisole concentrations were determined in duplicate
by a stability-indicating HPLC method at 0, 1, 2, 3, 4, 7, 14, 30, 60 and 90
days. The initial and final pHs of solutions were measured. Results: The
recovery of levamisole from tablets was 100 ± 2.1%. No color or odour changes were
observed during the study period. The oral solutions prepared from powder were
stable at least 90 days stored at 4 and 23°C. The oral solutions prepared from
tablets were stable at least 90 days at 4°C and 15 days when stored at 23°C.
The initial pH of solutions prepared from powder and tablets were 5.30 and
4.55, respectively. Initial and final pH values were significantly different
(p<0.001) for the two solutions. Conclusions: Levamisole 25 mg/mL
oral solutions can be prepared from tablets or powder with sterile water for irrigation
and stored for 90 days under refrigeration, taking account of the lack of
microbiological contamination.

Introduction

Treatment of recurrent idiopathic nephrotic syndrome
in children is often complicated by the toxicity of the therapeutic regimen
with corticosteroids, alkylating substances or cyclosporine. An alternative is
the treatment with levamisole, a potent anthelmintic compound with
immuno-modulating properties (1-3). In children, levamisole is given orally as
2 - 5 mg/kg daily or every other day depending on the patient response (4-6).
Levamisole is commercially available only in 50-mg tablets under the trade name
Ergamisol (Janssen Pharmaceutica). Levamisole is labelled by the United States Food
and Drug Administration (FDA) as adjuvant treatment
with fluorouracil but not labelled for the treatment of nephrotic
syndrome. No liquid dosage form of levamisole is available. An oral solution
would be highly desirable for children who are unable to swallow tablets and
allows the dose to be easily adjusted. Thus, an oral solution could be helpful
for levamisole administration. Few data exist on the levamisole stability in
liquid form. The purpose of this study was to determine the chemical stability
of levamisole in solution prepared from tablets and powder and stored at two
temperatures (4 and 23°C) in glass prescription bottles over 90 days period.

Methods

Formulation of levamisole
solutions

One hundred 50-mg levamisole tabletsa were
crushed to a fine powder in a glass mortar. Twenty mL of sterile water for
irrigationb were added and levigated into a paste. After 30 min, 100
mL of sterile water for irrigation were added and allowed to stand for 30 min.
Then, the milky suspension was filtered through a 7-µm filter of paperc
to exclude the insoluble excipients. Sterile waterb was added to the
clear solution in a volumetric flask to obtain a theoretical levamisole
concentration at 25mg/mL. The recovery of levamisole (n = 5)
obtained from compressed tablets was measured. Another solution of levamisole
25 mg/mL was prepared with levamisole pure powderd. The 5-g powder
was weighed and mixed with waterb in a 200 mL volumetric flask. Ten
millilitres from each levamisole solutions were transferred in 16 amber glass
prescription bottlese.

Storage of solutions

Six bottles of each formulation were stored at 4 ± 3°C and six
other bottles were stored at room temperature (23 ± 2°C) protected from light. Four bottles
were used to determine the stability study at 60 ± 5°C.

Sampling

From each
bottle, a sample (100 µL) was taken and mixed with 900 µL of waterb.
Then, 100 µL of this dilution was mixed with 300 µL of internal standard (1.5
mg/mL) and 600 µL of water in order to obtain a theoretical levamisole
concentration at 250 µg/mL. The diluted sample was assayed in duplicate by
high-performance liquid chromatography (HPLC), immediately after preparation
(day 0) and after 1, 3, 4, 7, 15, 30, 60 and 90 days. The apparent pH of each
solution was measured using a digital pH meterf at the beginning and
the end of the study. The appearance and color of the samples were assessed by
observing samples against black and white backgrounds, and any changes of odor
were noted at each time. The stability-indicating method was proven to ensure
that potential degradation products did not interfere with levamisole and
internal standard peaks.

HPLC Analysis

The HPLC method
was adapted from Vandamme et al (7).
The assay instrumentation required an isocratic pumpg, a manual
injectorh with a 20 µL loop, an ultraviolet light detectori
set at 235 nm, a C18 columnj set at room temperature and a recording
integratork. The mobile phase consisted of potassium hydrogen
phosphatel0.05 M
and acetonitrilem (85:15 v/v) used at a constant flow rate of 1
mL/min. Internal standard solution at 1.5 mg/mL was prepared by diluting
quinineo with waterb. Calibration curves were performed
with standard solution diluted in mobile phase to yield levamisole base concentrations
of 50, 100, 200, 300, 400 and 500 µg/mL, and 0.5 mg/mL of internal standard.
The standard curve (n = 5) was constructed by plotting the peak-height ratio of
levamisole to quinine against levamisole concentration, and was used for
calculating the drug concentration of the sample. The method was shown linear
(r>0.999) in the range of 50 to 500 µg/mL. Three levamisole base control
concentrations at 50, 200 and 500 µg/mL were analyzed to determine intra- and
inter-day coefficients of variation (CV). Each sample was injected in
duplicate. The repeatability was assessed with 6 determinations at 3 levels
(50, 200, 500 µg/mL) the same day. The intraday CVs were 3.79%, 1.18% and
0.63%, respectively. The reproducibility was assessed with 2 determinations on
3 days, at 3 levels (50, 200, 500 µg/mL). The interday CVs were 3.62%, 3.56%
and 3.69%, respectively. The limits of detection (signal/noise=3:1) and
quantification of levamisole were 0.36 and 1 µg/mL, respectively. The method
was shown to be stability-indicating by adjusting the pH of levamisole
solutions at 1 with acid (1.0
M HClp) and 12 with base (1.0 M NaOHq), by
oxidation with hydrogen peroxider and by storing oral solutions at 60°C. The stability
indicating assay proved degradation of levamisole and no interfering peak. The
retention times for levamisole and internal standard were 8 and 15 min,
respectively. The duration of chromatogram was approximately 20 min (Fig. 1).

Data analysis

The initial
concentration of levamisole was defined as 100%, and sample concentrations were
expressed as a percentage of the initial concentration remaining. The solution
was defined as being stable if the drug concentrations were not <90% of the
initial drug concentration. The significance of any difference between initial
and final pH values was evaluated by a Student’s t-test (α = 0.05).

Results

The
recovery of levamisole from tablets was 100 ± 2.1%. In the oral solution prepared from
powder, the mean concentrations of levamisole were >99% of the initial
concentration at 4°C and >96% of the initial concentration at 25°C for the 90-days
study period (Table 1). The mean concentrations of levamisole were >94% of
the initial concentration at 4°C and <64% of the initial concentration at
25°C over the study period for solution prepared from tablets (table 1). No
change of appearance, color or odor was noted with any of the solutions. The
apparent initial pH was significantly different between solutions prepared from
tablets and those prepared from the powder (Table 1). The final pH values of
solutions prepared from tablets stored at 4°C and 25°C increased significantly
(p <0.001) by 1.07 and 1.57 units, respectively, as compared with the
initial pH values. The final pH values of solutions prepared from powder stored
at 4°C and 25°C increased significantly (p <0.001) by 1.48 and 1.68 units,
respectively, as compared with the initial pH values.

Table
1. Stability of levamisole
solutions prepared from tablets and powder at 4 and 25°C.

Discussion

The complete
recovery of levamisole hydrochloride from tablets was related to its high water
solubility reported to be 210 g/L (8). Levamisole solutions prepared from
tablets and stored at 23°C appeared to be less stable than those prepared from
powder. Refrigeration storage was shown to permit a better stability of
levamisole solutions. Levamisole 25 mg/mL oral solutions prepared from powder
and tablets stored at 4°C were shown stable at least 90 days and could be then
used in clinical practice. This period of storage was conditioned by a possible
microbiological contamination. With lack of preservatives in solutions, a
period of 30 days stability seems more reasonable. Like temperature of storage,
pH had an impact on the chemical stability of levamisole. It was shown that the
rate of decomposition of levamisole rapidly increased between pH 5 and 7 and at
pH 8 it was about seventy times faster than at pH 2 (9-11). Excipients and pH
of solutions prepared from tablets could probably explain the difference in
stability. Thus, the use of the solution prepared from powder would be
preferable in practice. The taste of solutions was bitter and would be masked by
an additive.

Conclusion

An oral liquid
solution of levamisole at 25 mg/mL prepared from tablets or pure powder in
sterile water was shown chemically stable 90 days under refrigeration. These
oral solutions appear to be widely used as an alternative to the administration
of dry levamisole forms for the pediatric patients.